Method of separating biomolecules using hydrophobically-derivatized supports

ABSTRACT

A method of separating biomolecules in an aqueous mixture is disclosed comprising a obtaining a separation vessel containing separation media, wherein the separation media comprises a porous support with a hydrophobic monomer grafted thereon, the hydrophobic monomer having the structure: 
       CH 2 ═CR 4 C(O)NHC(R 1 R 1 )(C(R 1 R 1 )) n C(O)XR 3  
 
     wherein n is an integer of 0 or 1; R 1  is independently selected from at least one of: a hydrogen atom, alkyls, aryls, and alkylaryls, wherein the alkyls, aryls, and alkylaryls have a total of 10 carbon atoms or less; R 3  is a hydrophobic group selected from at least one of alkyls, aryls, alkylaryls and ethers, wherein the alkyls, aryls, alkylaryls and ethers have a total number of carbon atoms ranging from 4 to 30; R 4  is H or CH 3 ; and X is O or NH; wherein the hydrophobic monomer is derived from an amine or an alcohol (HXR 3 ) that has a hydrophilicity index of 25 or less; and
 
(b) passing the aqueous mixture through the separation vessel thereby separating the biomolecules. Such methods can be used to separate proteins, antibodies, fusion proteins, vaccines, peptides, enzymes, DNA, and/or RNA.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/213,572, filed Jul. 19, 2016, which is a continuation of U.S.application Ser. No. 13/258,018, filed Sep. 21, 2011, which is anational stage filing under 35 U.S.C. 371 of PCT/US2010/027978, filedMar. 19, 2010, which claims priority to U.S. Provisional Application No.61/165,132, filed Mar. 31, 2009, the disclosure of which is incorporatedby reference in its/their entirety herein.

TECHNICAL FIELD

The present disclosure generally relates to hydrophobic monomers, andtheir use, for example, in hydrophobically-derivatized supports. Thepresent disclosure also generally relates to the method of making andusing these hydrophobically-derivatized supports in applications such ashydrophobic interaction chromatography.

BACKGROUND

Hydrophobic interaction chromatography (HIC) is a chromatographytechnique based on the separation of molecules based on theirhydrophobicity. Generally, sample molecules in a high salt buffer areloaded on the HIC column. The salt in the buffer interacts with watermolecules to reduce the solvation of the sample molecules in solution,thereby exposing hydrophobic regions in the sample molecules, which areconsequently adsorbed by the stationary phase of the HIC column. Themore hydrophobic the molecule, the less salt needed to promote binding.Usually, a decreasing salt gradient is used to elute samples from thecolumn in order of increasing hydrophobicity. Sample elution may also beachieved by the addition of mild organic modifiers (e.g., solvents) ordetergents to the elution buffer, by changing the pH, or by the additionof chaotropic agents.

The HIC stationary phase typically comprises agarose, silica, or organicpolymer resins, which may be modified by hydrophobic ligands. One suchHIC stationary phase is prepared by reacting a hydrophobic ligandcomprising a nucleophile, to a particle (e.g., a bead) comprising anazlactone moiety. For example, U.S. Pat. No. 5,993,935 (Rasmussen etal.) describes the covalent bonding of azlactone moieties on the surfaceof particles with nucleophilic ligands by direct interaction (i.e.,without the need for an intermediate activation step).

U.S. Pat. No. 5,561,097 (Gleason, et al.) describes a method ofcontrolling the density of low molecular weight ligands, which arecovalently bonded to azlactone moieties on the surface of supports(e.g., particles). The density is controlled by conducting the covalentbonding reaction in the presence of a quencher. Theazlactone-functionalized support is typically prepared by polymerizationof an azlactone monomer or precursor to a support with subsequentcyclization to the azlactone. The azlactone-functionalized support isthen reacted with a ligand (such as benzyl amine) to produce aderivatized support. Although extremely low levels of side reactionssuch as hydrolysis take place during the course of the derivatizationreaction, some hydrolysis of the azlactone may indeed take place,generating carboxylic acid groups. When the end product is an ionexchange resin, this minor amount of side reaction is not a concern.However, in some applications, such as HIC stationary phases, thepresence of any ionic functionality, even trace amounts, can lead tochanges in performance, for example, in dynamic binding capacity and/orresolution.

HIC stationary phases are also susceptible to hydrolysis when exposed tobasic conditions if they are derived from hydrophobic (meth)acrylateesters and/or (meth)acrylamide monomers. For example, one molar sodiumhydroxide is often used to clean chromatography columns between uses,however these basic conditions can hydrolize the (meth)acrylate esterand/or (meth)acrylamide polymer. This hydrolysis leads to the formationof carboxylic acid functionality on the support, and thus to adegradation in chromatographic performance.

SUMMARY

In one aspect, the present disclosure provides filtration mediacomprising a porous support with a hydrophobic monomer grafted thereon,the hydrophobic monomer having the structure:

CH₂═CR⁴C(O)NHC(R¹R¹)(C(R¹R¹))_(n)C(O)XR³

wherein n is an integer of 0 or 1; R¹ is independently selected from:alkyls, aryls, and alkylaryls, wherein the alkyls, aryls, and alkylarylshave a total of 10 carbon atoms or less; R³ is a hydrophobic groupselected from: alkyls, aryls, alkylaryls and ethers, wherein the alkyls,aryls, alkylaryls and ethers have a total number of carbon atoms rangingfrom 4 to 30; R⁴ is H or CH₃; wherein the hydrophobic monomer is derivedfrom an amine (HNR³) that has a hydrophilicity index of 25 or less.

In another aspect, the present disclosure provides a compositioncomprising a hydrophobic monomer having the structure:

CH₂═CR⁴C(O)NHC(R²)₂(C(R¹R¹))_(n)C(O)NHR³

wherein n is an integer of 0 or 1; R¹ is independently selected from:alkyls, aryls, and alkylaryls, wherein the alkyls, aryls, and alkylarylshave a total of 10 carbon atoms or less; R² is selected from alkyls,aryls, and alkylaryls, wherein the alkyls, aryls, and alkylaryls have atotal of 10 carbon atoms or less; R³ is a hydrophobic group selectedfrom: alkyls, aryls, alkylaryls and ethers, wherein the alkyls, aryls,alkylaryls and ethers have a total number of carbon atoms ranging from 4to 30; R⁴ is H or CH₃; wherein the hydrophobic monomer is derived froman amine (HNR³) that has a hydrophilicity index of 25 or less.

In yet another aspect, method of separating biomacromolecules isdescribed, the method comprising:

contacting a solution comprising biomacromolecules to ahydrophobically-derivatized support, wherein thehydrophobically-derivatized support comprises a support grafted with ahydrophobic monomer of the following structure:

CH₂═CR⁴C(O)NHC(R¹R¹)(C(R¹R¹))_(n)C(O)XR³

wherein n is an integer of 0 or 1; R¹ is independently selected from:alkyls, aryls, and alkylaryls, wherein the alkyls, aryls, and alkylarylshave a total of 10 carbon atoms or less; R³ is a hydrophobic groupselected from: alkyls, aryls, alkylaryls and ethers, wherein the alkyls,aryls, alkylaryls and ethers have a total number of carbon atoms rangingfrom 4 to 30; R⁴ is H or CH₃; wherein the hydrophobic monomer is derivedfrom an amine (HNR³) that has a hydrophilicity index of 25 or less.

The terms “comprises” and variations thereof do not have a limitingmeaning where these terms appear in the description and claims.

The words “preferred” and “preferably” refer to embodiments of thedisclosure that may afford certain benefits, under certaincircumstances. However, other embodiments may also be preferred, underthe same or other circumstances. Furthermore, the recitation of one ormore preferred embodiments does not imply that other embodiments are notuseful, and is not intended to exclude other embodiments from the scopeof the disclosure.

The terms “a”, “an”, and “the” are used interchangeably with “at leastone” to mean one or more of the elements being described.

The term “and/or” means one or all of the listed elements or acombination of any two or more of the listed elements.

The term “alkyl” refers to a monovalent group that is a radical of analkane, which is a saturated hydrocarbon. The alkyl can be linear,branched, cyclic, or combinations thereof and typically has 1 to 30carbon atoms. In some embodiments, the alkyl group contains at least 1,2, 3, 4, 5, 6, 8, 10, 15, 20, or 25 carbon atoms; at most 30, 28, 26,25, 20, 15, 10, 8, 6, 5, 4, or 3 carbon atoms. Examples of alkyl groupsinclude, but are not limited to, methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, cyclohexyl, n-heptyl,n-octyl, and ethylhexyl.

The term “alkylene” refers to a divalent group that is a radical of analkane. The alkylene can be straight-chained, branched, cyclic, orcombinations thereof. The alkylene often has 1 to 30 carbon atoms. Insome embodiments, the alkylene group contains at least 1, 2, 3, 4, 5, 6,8, 10, 15, 20, or 25 carbon atoms; at most 30, 28, 26, 25, 20, 15, 10,8, 6, 5, 4, or 3 carbon atoms. The radical centers of the alkylene canbe on the same carbon atom (i.e., an alkylidene) or on different carbonatoms.

The term “aryl” refers to a monovalent group that is aromatic andcarbocyclic or heterocyclic. The aryl can have one to five rings thatare connected to or fused to the aromatic ring. The other ringstructures can be aromatic, non-aromatic, or combinations thereof andtypically has 1 to 30 carbon atoms. In some embodiments, the aryl groupcontains at least 1, 2, 3, 4, 5, 6, 8, 10, 15, 20, or 25 carbon atoms;at most 30, 28, 26, 25, 20, 15, 10, 8, 6, 5, 4, or 3 carbon atoms.Examples of aryl groups include, but are not limited to, phenyl,biphenyl, terphenyl, anthryl, naphthyl, acenaphthyl, anthraquinonyl,phenanthryl, anthracenyl, pyrenyl, perylenyl, and fluorenyl.

The term “alkylaryl” refers to a monovalent group that is a combinationof an alkyl and an aryl group. The alkylaryl can be an aralkyl, that is,an alkyl substituted with an aryl, or alkaryl, that is, an arylsubstituted with an alkyl. The alkylaryl can have one to five rings thatare connected to or fused to the aromatic ring and can comprise linear,branched, or cyclic segments, or combinations thereof. The alkylarylgroup typically has 1 to 30 carbon atoms. In some embodiments, thealkylaryl group contains at least 1, 2, 3, 4, 5, 6, 8, 10, 15, 20, or 25carbon atoms; at most 30, 28, 26, 25, 20, 15, 10, 8, 6, 5, 4, or 3carbon atoms.

The term “(meth)acrylamide” refers to compounds containing either anacrylamide or a methacrylamide structure or combinations thereof.Similarly, the term “(meth)acrylate” refers to compounds containingeither an acrylate or a methacrylate structure or combinations thereof.

The terms “polymer” and “polymeric material” refer to both materialsprepared from one monomer such as a homopolymer or to materials preparedfrom two or more monomers such as a copolymer, terpolymer, etc.Likewise, the term “polymerize” refers to the process of making apolymeric material that can be a homopolymer, copolymer, terpolymer, orthe like. The terms “copolymer” and “copolymeric material” refer to apolymeric material prepared from at least two monomers and includesterpolymers, quadpolymers, etc.

The terms “room temperature” and “ambient temperature” are usedinterchangeably to mean temperatures in the range of 20° C. to 25° C.

The above summary of the present disclosure is not intended to describeeach disclosed embodiment or every implementation of the presentdisclosure. The description that follows more particularly exemplifiesillustrative embodiments. In several places throughout the application,guidance is provided through lists of examples, which can be used invarious combinations. In each instance, the recited list serves only asa representative group and should not be interpreted as an exclusivelist.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts the hydrophobic interaction chromatograms (absorbanceversus elution volume) for Example 17 (Ex. 17) and Example 18 (Ex. 18).

FIG. 2 depicts the dynamic binding capacity chromatograms (absorbanceversus elution volume) for Examples 19 (Ex. 19) and Examples 20 (Ex. 20)and Comparative Example B (Comp. Ex. B) and Comparative Example C (Comp.Ex. C).

FIG. 3 depicts hydrophobic interaction chromatograms (absorbance versuselution volume) for Example 21 (Ex. 21) and Example 22 (Ex. 22).

FIG. 4 depicts a selected peak in a hydrophobic interaction chromatogram(absorbance versus elution volume) for Example 24 (Ex. 24) andComparative Example D (Comp. Ex. D).

DETAILED DESCRIPTION

There is a need to synthesize hydrophobic free-radically polymerizable(meth)acrylamide monomers that are easily synthesized and isolated.Additionally, there is a need to manufacture a polymer support thatcomprises hydrophobic character with reduced ionic character. There isalso a need to manufacture hydrophobic supports, which, for example, canbe used as an HIC stationary phase, that have both high dynamic bindingcapacity and good protein resolution characteristics.

This disclosure provides hydrophobic free-radically polymerizable(meth)acrylamide monomers. In some embodiments, these hydrophobic freeradically polymerizable (meth)acrylamide monomers can be polymerizedwith other monomers to produce hydrophobic supports. In someembodiments, these hydrophobic supports may be used to separatebiological and non-biological samples.

The hydrophobic monomers according to this disclosure have the structureaccording to formula (I):

CH₂═CR⁴C(O)NHC(R¹R¹)(C(R¹R¹))_(n)C(O)XR³  (I)

wherein n is an integer of 0 or 1; R¹ is independently selected from atleast one of a hydrogen atom, alkyls, aryls, and alkylaryls; R³ is ahydrophobic group selected from at least one of: alkyls, aryls,alkylaryls and ethers; R⁴ is H or CH₃; and X is O or NH.

In one embodiment, the hydrophobic monomer is derived from an amine oralcohol (HXR³) that has a hydrophilicity index of 25 or less.

In one embodiment, R¹ is independently selected from at least one of:hydrogen atoms, alkyls, aryls, and alkylaryls, wherein the alkyls,aryls, and alkylaryls have a total of 10 carbon atoms or less, 9 carbonatoms or less, 8 carbon atoms or less, 7 carbon atoms or less, 6 carbonatoms or less, 5 carbon atoms or less, 4 carbon atoms or less, or even 3carbon atoms or less. Examples of R¹ include: a hydrogen atom, a methylgroup, an ethyl group, and a phenyl group.

In one embodiment, R³ is a hydrophobic group selected from at least oneof: alkyls, aryls, alkylaryls and ethers, wherein the alkyls, aryls,alkylaryls and ethers have a total number of carbon atoms ranging from 4to 30. In some embodiments, the alkyls, aryls, alkylaryls and etherscontain at least 4, 5, 6, 8, 10, 12, 15, or 20 carbon atoms; at most 30,28, 26, 24, 20, 15, 12, 10, 8, or 6 carbon atoms. Examples of R³include: a benzyl group, a phenethyl group, a phenoxyethyl group, aphenylpropyl group, a butyl group, a pentyl group, a hexyl group, anoctyl group, a dodecyl group, an octadecyl group, and a phenylbutylgroup.

The hydrophobic monomers of this disclosure are synthesized at roomtemperature by a nucleophilic reaction between an alkenyl azlactone witha primary amine or alcohol ligand. The alkenyl azlactone includes5-member and 6-member azlactones with an alkenyl substituent, such asthose disclosed in formulas (II) and (III) below, wherein R¹ and R⁴ arethe same as those defined above.

Exemplary alkenyl azlactones include:4,4-dimethyl-2-vinyl-4H-oxazol-5-one (vinyldimethylazlactone),2-isopropenyl-4H-oxazol-5-one, 2-vinyl-4,5-dihydro-[1,3]oxazin-6-one,4,4-dimethyl-2-vinyl-4,5-dihydro-[1,3]oxazin-6-one,4,5-dimethyl-2-vinyl-4,5-dihydro-[1,3]oxazin-6-one, and combinationsthereof.

During the synthesis of the hydrophobic monomer, the primary amine oralcohol reacts with the carbonyl of the alkenyl azlactone, opening theazlactone ring and forming an adduct. The reaction solvent can beorganic (such as alcohols, ethers, hydrocarbons, esters, halogenatedsolvents, or combinations thereof), aqueous, or mixed, but should becapable of dissolving or at least partially dissolving the alkenylazlactone and the primary amine or alcohol ligand. Although theazlactone moiety is quite stable towards hydrolysis, it is known thatring opening by water can occur as a minor side-reaction. Thishydrolysis can lead to the formation of carboxyl groups, which mayimpart ionic character. Therefore, in one embodiment, the covalentbonding of the alkenyl azlactone with the primary amine or alcoholligand is conducted in an organic solvent to ensure little to nohydrolysis of the alkenyl azlactone.

The azlactone moiety reacts rapidly with the primary amine or alcohol ofthe ligand forming a direct covalent bond with no displacement of aby-product molecule. Thus, purification of the resulting hydrophobicmonomer is minimized. Typically, the hydrophobic monomer precipitatesfrom the reaction solvent in very pure form (for example, greater than90% purity, or even greater than 99% purity) and can be isolated bysimple filtration and drying. Optionally, the hydrophobic monomer can berecrystallized to further enhance its purity, although this is generallynot necessary.

For purposes of this disclosure, the selection of the primary amine oralcohol ligand utilized in the synthesis of the hydrophobic monomer willdetermine the hydrophobicity of the resulting hydrophobic monomer. Theprimary amine or alcohol ligand comprises a hydrophobic group selectedfrom at least one of: alkyls, aryls, alkylaryls and ethers, wherein thealkyls, aryls, alkylaryls and ethers have a total number of carbon atomsranging from 4 to 30. In some embodiments, the alkyls, aryls, alkylarylsand ethers contain at least 4, 5, 6, 8, 10, 12, 15, or 20 carbon atoms;at most 30, 28, 26, 24, 20, 15, 12, 10, 8, or 6 carbon atoms.

In one embodiment, the hydrophobic monomer has a calculatedhydrophilicity index (HI) of 25 or less, 20 or less, 15 or less, or even10 or less. The HI is an empirical concept that is described in detailin U.S. Pat. No. 4,451,619 (Heilmann, et al.), herein incorporated byreference. In general, this concept allows one to determine the effectthat an added primary amine or alcohol ligand will have on thehydrophilicity or hydrophobicity of the final product, (i.e., ahydrophobic monomer, a polymerizable mixture, or ahydrophobically-derivatized support). For purposes of this disclosure,HI is calculated based on the primary amine or alcohol ligand of R³(i.e., HXR³). The HI of the hydrophobic monomer according to thisdisclosure is defined as:

HI=total molecular weight of the hydrophilic groups in HXR³/molecularweight of HXR³×100

The hydrophilic groups are generally those that are functionally capableof forming hydrogen bonds with water. Examples of hydrophilic groupsinclude: —N—, —NH—, —NH₂, —OH, —O—, C═O, —CO₂H, —CO₂ ⁻M⁺ (where M⁺ is analkali or alkaline earth metal ion), —SO₃H, —SO₃ ⁻M⁺, —CONH2, —SH, —NR₃⁺X⁻ (where R═C₁₋₄ alkyl and X⁻ is typically a halide), —NHCONH—, and thelike.

Primary amine or alcohol ligands that tend to impart a hydrophiliccharacter to final product typically have an HI of greater than 30,while the primary amine or alcohol ligands that impart a hydrophobiccharacter typically have an HI of less than 20. Primary amine or alcoholligands with an HI between 20 and 30 are typically classified as“neutral” or “borderline”.

Table 1 lists the HI of a number of primary amine and alcohol ligandsthat have been found to be useful for the purposes of this disclosure.Interestingly, some ligands having a “borderline” HI (for example,butylamine and phenoxyethylamine) can be used in applications such asprotein purification.

TABLE 1 Hydrophilicity Index for Primary Amine Ligands Total HydrophilicMolecular Component Ligands weight MW HI Benzylamine 107 16 15Phenethylamine 121 16 13 Phenoxyethylamine 137 32 23 Phenylpropylamine135 16 12 Phenylbutylamine 149 16 11 Butylamine 73 16 22 Hexylamine 10116 16 Octylamine 129 16 12 Octadecylamine 270 16 6 Phenylbutanol 150 1711

In some applications, hydrophobic monomers derived from primary amineligands are preferred over those derived from alcohols due to thepresence of two amide functional groups, which result in a lowersusceptibility towards hydrolysis.

Exemplary hydrophobic monomers include:CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)NH(CH₂)₄C₆H₅;CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)O(CH₂)₄C₆H₅;CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)NHCH₂C₆H₅;CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)NH(CH₂)₂C₆H₅;CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)NH(CH₂)₂OC₆H₅;CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)NH(CH₂)₃C₆H₅;CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)NH(CH₂)₃CH₃;CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)NH(CH₂)₅CH₃;CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)NH(CH₂)₇CH₃;CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)NH(CH₂)₁₁CH₃;CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)NH(CH₂)₁₇CH₃; or combinations thereof.

The hydrophobic monomers of this disclosure may have a tendency insolution to self-associate or, when in the presence of other monomers orpolymers, associate with the other monomers or polymers. Thisassociation can be the result of two separate interactions. First, thehydrophobic groups of the hydrophobic monomers may associate with oneanother, or with the other monomers or polymers, especially in aqueousmedia. Secondly, hydrogen-bonding interactions can occur between theamide functionalities of the hydrophobic monomers or between thehydrophobic monomers and the other monomers or polymers. Thehydrogen-bonding interactions are particularly prevalent with thehydrophobic monomers derived from amine ligands, wherein two amidegroups are present. The tendency of the hydrophobic monomers toassociate may be advantageous, for example, enabling one to control apolymer microstructure (such as, for example, the distribution ofhydrophobic groups within a polymerized support) and/or the propertiesof the final product (such as, for example, hydrophobicity and/orviscosity) by manipulating the polymerization conditions.

In addition to the hydrophobic portion, which can participate inhydrophobic interactions, the hydrophobic monomer also comprises anunsaturated site (e.g., a double bond), which is derived from thealkenyl substituent of the alkenyl azlactone. In one aspect of thisdisclosure, this site of unsaturation enables the hydrophobic monomer toparticipate in free radical polymerization schemes. Thus, thesehydrophobic monomers may be added to a polymerizable mixture, which thenmay be used to make a hydrophobically-derivatized support.

In one aspect, the polymerizable mixture comprises the hydrophobicmonomer.

In one embodiment, the polymerizable mixture further comprises across-linking monomer. The cross-linking monomer comprises a pluralityof polymerizable groups, which during polymerization, extend the chainlength of the polymer backbone and during curing, physically join (orcross-link) the polymer backbones. Cross-linking aids in the mechanicalstability of the resulting article.

The cross-linking monomers include, for example,N,N′-alkylenebis(meth)acrylamides, alkylenebis(meth)acrylates,divinylaromatics, polyallylesters or combinations thereof. Exemplarycross-linking monomers include: ethylenically unsaturated esters such asethylene diacrylate, ethylene dimethacrylate, trimethylolpropanetriacrylate and trimethacrylate; and α- and β-unsaturated amides, suchas methylene bis(acrylamide), methylene bis(methacrylamide),N,N′-diacryloylpiperazine, N,N′-diacryloyl-1,2-diaminoethane, andN,N′-dimethacryloyl-1,2-diaminoethane; or combinations thereof. In someapplications, such as HIC-type applications, theN,N′-alkylenebis(meth)acrylamides are preferred due to theirhydrophilicity and increased hydrolytic stability.

In one embodiment, the polymerizable mixture further comprises anon-cross-linking monomer. The non-cross-linking monomer is used topropagate the polymer backbone (i.e., extend the chain length), but doesnot generally participate in physically joining polymer backbones or maybe used to solubilize the hydrophobic monomer. In one embodiment, thenon-cross-linking monomers are uniformly distributed throughout thehydrophobically-derivatized support and assist in uniformly distributingthe hydrophobic monomers in the hydrophobically-derivatized support.

The non-cross-linking monomers include: (meth)acrylate, (meth)acrylamidemonomers, or combinations thereof. Exemplary non-cross-linking monomersinclude: dimethylacrylamide, acrylamide, methacrylamide,hydroxyethyl(meth)acrylate, or combinations thereof. The use of thesenon-cross-linking monomers can provide significant enhancements to theproperties of the hydrophobically-derivatized support. For example,although not wanting to be bound by theory, the concentration and typeof non-cross-linking monomers are thought to influence the porosity ofthe hydrophobically-derivatized support and/or the distribution of thehydrophobic monomer.

In one embodiment, the hydrophobic monomers of formula (I) andnon-crosslinking monomers may be used in the preparation ofhydrophobically-associating polymers, which are useful as aqueous fluidrheology or flow modifiers. In one embodiment, thesehydrophobically-associating polymers may be used, for example, asflocculation aids for waste water treatment and dewatering sludge, andfor rheology control for secondary and tertiary oil recovery. In anotherembodiment, these hydrophobically-associating polymers may also be usedas separation media for capillary electrophoresis in DNA or RNAsequencing and separations.

The amount of hydrophobic monomer, cross-linking monomer, and/ornon-cross-linking monomer may be important in the properties of thepolymerized mixture and the resulting hydrophobically-derivatizedsupport. Generally, the amount of hydrophobic monomer added controls thehydrophobicity of the resulting hydrophobically-derivatized support. Thehydrophobic monomer can be added at 0.1 to 30% by weight relative to thetotal monomer amount. In some embodiments, the hydrophobic monomer is atleast 0.1, 0.2, 0.5, 1, 1.5, 3, 5, 10, 15, 20, or 25% by weight; at most30, 25, 20, 15, 10, 5, 3, 1.5, 1, 0.5% by weight relative to the totalmonomer amount. Generally, the amount of cross-linking monomer addedcontrols the rigidity and swelling ability of the particle. Thecross-linking monomer can be added at 0-99.9% by weight relative to thetotal monomer amount. In some embodiments, the cross-linking monomer isat least 0, 0.1, 0.5, 1, 1.5, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90,95, or 98% by weight; at most 99.9, 99.5, 99, 98, 95, 90, 80, 75, 70,60, 50, 40, 30, 20, 10, 5, 3, 1, or 0.5% by weight relative to the totalmonomer amount. Generally, the amount of non-cross-linking monomer aidsin the determination of final copolymer properties, includinghydrophilicity, solubility, porosity, crosslink density, rigidity, etc.,depending upon the final application. The non-cross-linking monomer maybe added at 0-99.9% by weight relative to total monomer amount. In someembodiments, the non-cross-linking monomer is at least 0, 0.1, 0.5, 1,1.5, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or 98% by weight; atmost 99.9, 99.5, 99, 98, 95, 90, 80, 75, 70, 60, 50, 40, 30, 20, 10, 5,3, 1, or 0.5% by weight relative to the total monomer amount.

In one embodiment, the amount of the cross-linking monomer is greaterthan the amount of the non-cross-linking monomer. In one embodiment, theamount of the non-cross-linking monomer is greater than the amount ofthe hydrophobic monomer. In yet another embodiment, the amount of thecross-linking monomer is greater than the amount of thenon-cross-linking monomer, which is greater than the amount of thehydrophobic monomer. For example, in an HIC application, the amount ofcross-linking monomer is 60% or more by weight relative to the totalmonomer amount to enable rigidity of the hydrophobically-derivatizedsupport to withstand the pressure tolerances, and the amount ofhydrophobic monomer is 10% or less by weight relative to the totalmonomer amount to ensure release of the analyte from the HIC stationaryphase.

In another embodiment, the polymerizable mixture may comprise a porogen.A porogen may be added to the polymerizable mixture to control the porestructure of the hydrophobically-derivatized support, especially whenthe hydrophobically-derivatized support is a particle or a coating.

Pore formation or porosity in polymeric materials is described in detailby Sherrington, Chem. Commun., 2275-2286 (1998). With some materials,especially gel-type materials, porosity is formed during thepolymerization or curing process as a result of the entanglement and/orcrosslinking of the polymer chains. Typically this porosity is very lowor nonexistent unless the polymer network is highly swollen by asolvent. Alternately, porogens can be added to the composition to createpermanent pores. Added porogens typically influence the timing of phaseseparation of the forming polymer network from the rest of the monomerphase mixture. Examples of porogens include: water, alcohols (such as,for example, methanol, ethanol, and isopropanol), ethylene glycol,propylene glycol, polyols having at least three hydroxy groups (such as,for example, glycerol, inositol, glucose, sucrose, maltose, dextran,pentaerithritol, trimethylolethane, timethylolpropane,dipentaerithritol, and tripentaerithritol), and polymeric porogens (suchas, for example, polyethylene glycol, polypropylene glycol, polyacrylicacid, polysaccharide, and the like), dispersed organic aggregate (suchas, for example, ethoxylated hydrocarbons), or combinations thereof.

Other factors may also be important in controlling the pore structure ofthe hydrophobically-derivatized support including, for example, theinteraction between the co-monomer composition and the selection of theporogen(s), the mass ratio between the non-cross-linking monomer and thecross-linking monomer, the chemical structure of the non-cross-linkingmonomer, or combinations thereof.

As mentioned above, the polymerizable mixture may be used to form ahydrophobically-derivatized support. This hydrophobically-derivatizedsupport may be obtained by at least one of: graft polymerizing thepolymerizable mixture onto a substrate, coating the polymerizablemixture onto a substrate and polymerizing the polymerizable mixture onthe surfaces of the substrate, or polymerizing and cross-linking thepolymerizable mixture to form particles, which may be used as supportsthemselves or may added to other porous substrates.

The degree of hydrophobicity of the hydrophobically-derivatized supportis controlled by the nature of the hydrophobic ligand, the amount ofhydrophobic ligand present on the support surface, and/or thedistribution of the hydrophobic groups on the support surface (which inthe case of hydrophobically-derivatized particles, is controlledprimarily by pore structure and swell volume of the particle).

In one embodiment, the surface of a pre-existing support is exposed tohigh energy radiation to generate free radical reaction sites on thesurface as disclosed in U.S. Pat. No. 5,344,701 (Gagnon et al.).Exposure of the pre-existing support with the polymerizable mixture cantake place simultaneously with or subsequent to the irradiation of thepre-existing support. Depending on the type of radiation and otherprocess conditions, the polymerizable mixture can either be grafted tothe surface of the pre-existing support or can be formed as a coating onthe pre-existing support or can become particles enmeshed within voidspaces of the support. In the former instance, the hydrophobic monomeris covalently bound to the pre-existing support. The pre-existingsupport may be treated with plasma, corona, beta, gamma, electron-beam,x-ray, ultraviolet, and other electromagnetic radiation as is known inthe art. The radiation may occur in the presence of other compounds,such as, for example, oxygen, or photoinitiators. The pre-existingsupports, depending on the final use, may be porous or non-porous,continuous or non-continuous, and flexible or inflexible. Examples ofpre-existing supports may include: woven webs, nonwoven webs, fibrouswebs, microporous membranes, fibers, hollow fibers, tubes, microporousfilms, nonporous films, or combinations thereof. The pre-existingsupports may be made from a variety of materials including ceramics,glass, metallic, polymeric materials, or combinations thereof. Examplesof suitable polymeric materials include: polyalkylenes such aspolyethylene and polypropylene; halogenated polymers such as polyvinylchloride and polyvinylidene fluoride; polyamides such as nylons;polystyrenes; poly(ethylene vinyl acetate); polyacrylates such aspolymethyl methacrylate; polycarbonate; cellulosics such as celluloseacetate butyrate; polyesters such as poly(ethylene teraphthalate); polyimidines; polyurethanes; or combinations thereof.

In another embodiment, the polymerizable mixture is coated onto thesurface of a pre-existing support. Typically, the alkenyl moiety of thehydrophobic monomer is not covalently bound to the surface of thepre-existing support, therefore, the polymerizable mixture alsocomprises a cross-linking monomer and optionally a non-cross-linkingmonomer to cross-link the polymerizable mixture onto the pre-existingsupport. The pre-existing supports are similar to those described above.The polymerization and resultant cross-linking may be initiated bychemical and/or physical means including, for example, redox chemistry,thermal initiation, UV irradiation or by ionizing radiation (such as,for example, e-beam and gamma radiation), or by other means as is wellknown in the art.

In one embodiment, the hydrophobic monomer is polymerized to producehydrophobically-derivatized particles. The hydrophobic monomer, across-linking monomer, and optionally a non-cross-linking monomer aremixed together and polymerized as an inverse suspension. As is apparentto one skilled in the art, the initiation system, suspending medium,stirring rate and the suspending agent are all essentially independentand important variables in the polymerization process. In oneembodiment, the monomers are dissolved in a water/alcohol solution, thissolution is suspended as droplets in an organic, immiscible medium, andsodium persulfate and tetramethylethylenediamine are used to initiatethe polymerization. Substitution of the various components by comparablematerials can certainly be made, and such substitutions would not beoutside the spirit and scope of the present disclosure.

The hydrophobically-derivatized particles of this disclosure can have aspherical shape, a regular shape, or an irregular shape. Size of theazlactone-derived functionalized particles can vary widely within thescope of the disclosure. Generally the size of the azlactone-derivedfunctionalized particles ranges from 0.1 micrometer (μm) to 5millimeters (mm) in average diameter.

In one embodiment, the hydrophobically-derivatized particles areconfined. For example, the hydrophobically-derivatized particles can beplaced in a vessel (such as a tube), enclosing at least one end of thevessel with a frit to create a chromatographic column. Suitable columnsare known in the art and can be constructed of such materials as glass,polymeric material, stainless steel, titanium and alloys thereof, ornickel and alloys thereof. Methods of filling the column to effectivelypack particles in the column are known in the art. The chromatographiccolumn, when packed with the hydrophobically-derivatized particles, canbe used in HIC applications.

Although the average particle size in chromatography can be as large as2000 micrometers, the average particle size is typically no greater than500 micrometers. If the average particle size is larger than about 500micrometers, the efficiency of the chromatographic process may be low,especially for the purification or separation of large biomacromoleculessuch as proteins that often have low diffusion rates into the pores ofchromatographic particles.

In another embodiment, the hydrophobically-derivatized particles aredispersed within a continuous, porous matrix. The continuous, porousmatrix is typically at least one of a woven or non-woven fibrous web,porous fiber, porous membrane, porous film, hollow fiber, film, or tube.Suitable continuous, porous matrixes are further described in U.S. Pat.No. 5,993,935 (Rasmussen et al.).

In yet another embodiment, the hydrophobically-derivatized particles aredisposed on a surface of a filtration medium. The filter element can bepositioned within a housing to provide a filter cartridge. Suitablefiltration medium and systems that include a filter cartridge arefurther described, for example, in U.S. Pat. No. 5,468,847 (Heilmann etal.). Such a filter cartridge can be used, for example, to purify orseparate biomolecules. Typically, less rigid particles or smaller porousparticles can be utilized within a filter cartridge compared to within achromatographic column due to the lower pressure drops inherent in thefilter cartridge system.

In one aspect of the present disclosure, the hydrophobically-derivatizedsupports have a reduced amount of ionic groups at the surface of thehydrophobically-derivatized support. Although, not wanting to be boundby theory, it is believed that the hydrophobically-derivatized supportsare able to be prepared with even less ionic functionality than themethods currently known in the art. Less ionic functionality on thesupport surface is thought to be a result of the hydrophobic monomersbeing prepared and purified prior to any contact with water (i.e.,conducting the synthesis of the hydrophobic monomer in organic solventwith no hydrolysis), and conducting the polymerization reactions withmonomers that are resistant to hydrolysis.

Azlactones are known to be susceptible to attack by water, which canlead to the formation of carboxyl groups, which may change theselectivity of the surface, imparting both ion-exchange and hydrophiliccharacter to the support's surface. In the present disclosure, theazlactone ring may be opened and covalently bonded to the hydrophobicligands under non-aqueous conditions, which may limit the generation ofionic groups at the surface of the support. By having fewer competingside reactions, a more pure hydrophobic support can be generated (i.e.,less ion-exchange character exhibited by the hydrophobically-derivatizedsupport). Thus, the hydrophobically-derivatized supports of the presentdisclosure can be more sensitive in hydrophobic interactions and not beinfluenced by other functional group interactions (e.g. ionic). Thisprovides a more specific separation that is based only on hydrophobicinteractions.

Further, the hydrophobically-derivatized supports of the presentdisclosure display hydrophobicities that are comparable to prior artmaterials, but comprise much lower hydrophobic ligand densities. For thepurposes of this discussion, “ligand density” means micromoles of ligandper milliliter of packed support material. While not wanting to be boundby theory, it is believed that the hydrophobically-derivatized supportsof the present disclosure display a more random and even distribution ofthe hydrophobic ligands on the surface of the support, thus leading to amore efficient utilization of the hydrophobic ligand for interactionwith the analyte (e.g., protein) of interest.

In one embodiment, neither the polymerizable mixture nor thehydrophobically-derivatized support comprises a quencher. Because thehydrophobic monomer comprises the hydrophobic ligand covalently bondedto the azlactone, a quencher, such as described in U.S. Pat. No.5,561,097 (Gleason, et al.) is not needed when forming thehydrophobically-derivatized supports.

Due to the hydrophobic nature of the hydrophobically-derivatizedsupports, the hydrophobically-derivatized supports may be used for thepurification of biological materials, for example, proteins, antibodies,fusion proteins, vaccines, peptides, enzymes, DNA, RNA, or combinationsthereof, as well as non-biological molecules with hydrophobiccharacteristics, in applications such as HIC.

The hydrophobically-derivatized supports of the present disclosure mayhave advantages over prior art HIC supports. In the present disclosure,an alkenyl azlactone is reacted with a hydrophobic ligand comprising anucleophile, for example an amine, to form the hydrophobic monomer (oradduct). By using the hydrophobic ligand as part of the hydrophobicmonomer in the polymerization step, instead of adding the hydrophobicligand to a support already comprising an attached azlactone group (suchas disclosed in U.S. Pat. Nos. 5,993,935 and 5,561,097), thehydrophobically-derivatized supports of the present disclosure may beachieved with improved uniformity in the distribution of the hydrophobicligand over the surface of the support and with fewer ionic sitespresent at the surface of the support. These characteristics may becritical in some HIC applications. The hydrophobically-derivatizedsupports of the present disclosure may be more resistant to hydrolysisthan HIC supports prepared by polymerizing hydrophobic (meth)acrylateesters and/or (meth) acrylamide monomers due to the steric hindranceprovided by the —C(R¹R¹) group interposed between the two carbonylgroups.

EXAMPLES

The following examples are merely for illustrative purposes and are notmeant to limit in any way the scope of the appended claims. All parts,percentages, ratios, and the like in the examples are by weight, unlessnoted otherwise. All raw materials are commercially available or knownto those skilled in the art unless otherwise stated or apparent. Thestructures of all novel hydrophobic monomers were confirmed by H′nuclear magnetic resonance and infrared spectroscopy.

Materials

Table of Materials Name Description Vinyl Purchased from SNPE, Inc.,Princeton, N.J. dimethylazlactone N- 3M, prepared by the proceduredescribed in acryloylmethylalanine U.S. Pat. No. 4,304,705 (Heilmann, etal.) PEG 2,000 A polyethylene glycol having a molecular weight of1800-2200 g/mole commercially available from Merck Schuchadt OHG,Hohenbrunn, Germany. PEG 6000 A polyethylene glycol having a molecularweight of 5000-7000 g/mole commercially available from Merck SchuchadtOHG, Hohenbrunn, Germany. PEG 10,000 A polyethylene glycol having amolecular weight of 9000-11250 g/mole commercially available from MerckSchuchadt OHG, Hohenbrunn, Germany.

Preparation of Hydrophobic Monomers

Example 1: The following procedure was used to prepare a4-phenylbutylamine/VDM adduct. Methyl-tert-butyl ether (MTBE, 100 ml(milliliter)) was added to a 1,000 ml, 3-necked flask, equipped with acondenser, overhead mixing paddle at 400 rotations per minute (rpm), andnitrogen inlet, in an ice bath. Vinyldimethylazlactone (VDM, 10.44 g(gram)) was added to the flask. 4-Phenylbutylamine (10 g) was added toan addition funnel. The bottle in which the 4-phenylbutylamine wasstored before addition to the addition funnel was rinsed with portionsof MTBE (10 ml total). The MTBE used to rinse the bottle was added tothe addition funnel. The 4-phenylbutylamine was added drop-wise over10-minutes to the flask that contained the VDM. After addition of the4-phenylbutylamine to the VDM was completed, the addition funnel wasrinsed with 10 ml of MTBE. A white precipitate (product) formed almostimmediately. The reaction was then allowed to proceed with mixing, undernitrogen, at 0° C. for 60 minutes.

The flask and at least 300 ml of MTBE were transferred to a freezer setat about −20° C. Crystallization of the white precipitate (product) wasallowed to occur for 1 to 2 hours. The solid product was filtered on afritted funnel with three 100 ml washes of the chilled MTBE. The productwas dried overnight in a vacuum oven at 60° C. and about 25 inches Hgvacuum. The % yield was about 90%. The purity was checked by silica thinlayer chromatography (TLC) with MTBE as the mobile phase. Both of thereactants were much more soluble in MTBE than the product. The meltpoint of the product, or adduct, was 89-91° C.

Example 2: The following procedure was used to prepare a benzylamine/VDMadduct. A similar procedure as that described for Example 1 above wasfollowed except 49.3 g of benzylamine (instead of 4-phenylbutylamine),69.9 g of VDM, and 393 ml of diethyl ether (instead of MTBE) were used.The % yield was about 84%.

Example 3: The following procedure was used to prepare aphenethylamine/VDM adduct. A similar procedure as that described forExample 1 above was followed except 43.8 g of phenethylamine (instead of4-phenylbutylamine), 36.3 g of VDM, and 300 ml of MTBE were used. The %yield was about 94%.

Example 4: The following procedure was used to prepare aphenoxyethylamine/VDM adduct. A similar procedure as that described forExample 1 above was followed except 10 g of phenoxyethylamine (insteadof 4-phenylbutylamine), 10.6 g of VDM, and 150 ml of MTBE were used. The% yield was about 92%.

Example 5: The following procedure was used to prepare a3-phenylpropylamine/VDM adduct. A similar procedure as that describedfor Example 1 above was followed except 24.7 g of 3-phenylpropylamine(instead of 4-phenylbutylamine), 24.4 g of VDM, and 150 ml of MTBE wereused. The % yield was about 77%.

Example 6: The following procedure was used to prepare a butylamine/VDMadduct. A similar procedure as that described for Example 1 above wasfollowed except 43.9 g of butylamine (instead of 4-phenylbutylamine),83.4 g of VDM, and 500 ml of diethylether (instead of MTBE) were used.The % yield was greater than 85%.

Example 7: The following procedure was used to prepare an octylamine/VDMadduct. A similar procedure as that described for Example 1 above wasfollowed except 77.6 g of octylamine (instead of 4-phenylbutylamine),83.4 g of VDM, and 650 ml of diethylether (instead of MTBE) were used.The % yield was greater than 85%.

Example 8: The following procedure was used to prepare adodecylamine/VDM adduct. A similar procedure as that described forExample 1 above was followed except 26.9 g of dodecylamine (instead of4-phenylbutylamine), 13.9 g of VDM, and 250 ml of diethylether (insteadof MTBE) were used. The % yield was greater than 85%.

Example 9: The following procedure was used to prepare anoctadecylamine/VDM adduct. A similar procedure as that described forExample 1 above was followed except 26.95 g of octadecylamine (insteadof 4-phenylbutylamine), 13.9 g of VDM, and 250 ml of diethylether(instead of MTBE) were used. The product was isolated by evaporating thediethylether solvent, and the % yield was greater than 95%.

Example 10: The following procedure was used to prepare a hexylamine/VDMadduct. A similar procedure as that described for Example 1 above wasfollowed except 60.7 g of hexylamine (instead of 4-phenylbutylamine),83.4 g of VDM, and 675 of ml diethylether (instead of MTBE) were used.The % yield was greater than 85%.

Example 11: The following procedure was used to prepare a4-phenyl-1-butanol/VDM adduct. Heptane (50 ml) was added to a 250 mlround bottom flask, equipped with a condenser and magnetic stir bar, inan ice bath. 4-Phenylbutanol (5.00 g) was added to the flask, followedby 5 drops of diazabicycloundecene (DBU) as catalyst. VDM (5.00 g) wasadded to an addition funnel along with heptane (25 ml). The contents ofthe dropping funnel were added dropwise over 5 minutes to the flask.After the addition was completed, the mixture was stirred for 45minutes, then the ice bath was removed. A colorless oil (product) hadformed. The reaction was then allowed to proceed with mixing for anadditional 2 hours. The heptane supernate was poured off, additionalheptane (50 ml) was added, the mixture was stirred another 15 minutes,then left to stand with no stirring for 10 minutes. The heptanesupernate again was poured off, and the residual oil was stripped on arotary evaporator with heating at 35° C. to give 9.28 g of colorless oil(96.5% yield), which crystallized at room temperature.

Preparation of Particles

Example 12: Heptane (174 ml) and 1.4 ml of polymer stabilizer solution((0.1 g of a polymer comprising a ratio of 92.5 isooctylacrylate to 7.5VDM, which has been ring opened with ammonia) per ml of toluene) wereadded to a 1 L Mortonized round bottom flask equipped with an overheadstirrer, thermocouple, reflux condenser, and nitrogen gas inlet. Theoverhead stirrer was adjusted to a stir rate of approximately 300 rpmand the reaction flask was heated to 35° C. under a slow nitrogen gaspurge. Methylene bis-acrylamide (MBA, 11.31 g), 2.09 g of acrylamide(AAm), and 0.60 g of 4-phenylbutylamine/VDM adduct (PhBVDM, preparedaccording to Example 1 above) were added to a 250 ml Erlenmeyer flaskequipped with a stir bar. Isopropyl alcohol (62.5 ml) and 42 ml of waterwere added to dissolve the solids. 10 g of a 50% aqueous solution of PEG2,000 was then added. Upon dissolution of all solids, sodium persulfatewas added to the stirred solution (0.56 g in 3 ml water). The aqueousphase was added to the organic phase and mixed until the reactionmixture reached 35° C. Tetramethylethylenediamine (0.55 ml) was added toinitiate the polymerization. The polymerization reaction was stirred for2 hours while particles formed.

The particles were course filtered and washed twice with acetone (250 mleach), twice with methanol (250 ml each), and then twice with acetone(250 ml each). The resulting particles were transferred to a 500 mlErlenmeyer flask. Acetone (300 ml) was added to suspend the particles.The suspended particles were sonicated for approximately 15 minutes,then filtered. The resulting particles were classified to a meanparticle size of approximately 60 μm (micrometers) using a series ofstacked sieves.

Example 13: A similar procedure as that described in Example 12 wasfollowed except PEG 6,000 was added as a porogen additive instead of PEG2,000.

Example 14: Heptane (348 ml) and 2.8 ml of polymer stabilizer solutionwere added to a 1 L Mortonized round bottom flask equipped with anoverhead stirrer, thermocouple, reflux condenser, and nitrogen gasinlet. The overhead stirrer was adjusted to a stir rate of approximately300 rpm and the reaction flask was heated to 35° C. under a slownitrogen gas purge. MBA (21.36 g), 5.50 g of dimethylacrylamide (DMA),and 1.14 g of PhBVDM were added to a 250 ml Erlenmeyer flask equippedwith a stir bar. 125 ml of isopropyl alcohol and 84 ml of water wereused to dissolve the solids. 20 g of a 50% aqueous solution of PEG 6,000was then added. Upon dissolution of all solids, sodium persulfate wasadded to the stirred solution (1.10 g in 6 ml water). The aqueous phasewas added to the organic phase and mixed until the reaction reached 35°C. Tetramethylethylenediamine (1.10 ml) was added to initiate thereaction. The polymerization reaction was stirred for 2 hours untilparticles were formed.

The particles were course filtered and washed twice with acetone (250 mleach), twice with methanol (250 ml each), and twice with acetone (250 mleach). The resulting particles were transferred to a 500 ml Erlenmeyerflask. Acetone (300 ml) was added to suspend the particles. Thesuspended particles were sonicated for approximately 15 minutes, thenfiltered. The resulting particles were classified to a mean particlesize of approximately 60 μm using a series of stacked sieves.

Example 15: A similar procedure as that described in Example 14 wasfollowed except PEG 10,000 was added as a porogen additive instead ofPEG 6,000.

Example 16: Heptane (348 ml), toluene (188 ml) and 1.4 ml of polymerstabilizer solution were added to a 1 L Mortonized round bottom flaskequipped with an overhead stirrer, thermocouple, reflux condenser, andnitrogen gas inlet. The overhead stirrer was adjusted to a stir rate ofapproximately 360 rpm and the reaction flask was heated to 35° C. undera slow nitrogen gas purge. MBA (12.89 g) and 1.11 g of benzylamine/VDMadduct (prepared according to Example 2 above) were added to a 250 mlErlenmeyer flask equipped with a stir bar. Isopropyl alcohol (65 ml) and47 ml of water were added to dissolve the solids. Ethylene glycol (25ml) was then added. Upon dissolution of all solids, sodium persulfate(0.55 g in 3 ml water) was added to the stirred solution. The aqueousphase was added to the organic phase and mixed until the reactionreached 35° C. Tetramethylethylenediamine (0.55 ml) was added toinitiate the reaction. The polymerization reaction was stirred for 2hours until beads formed.

The particles were course filtered and washed twice with acetone (250 mleach), twice with methanol (250 ml each), and then twice with acetone(250 ml each). The resulting particles were transferred to a 500 mlErlenmeyer flask. Acetone (300 ml) was added to suspend the particles.The suspended particles were sonicated for approximately 15 minutes, andthen filtered. The resulting particles were classified to a meanparticle size of approximately 65 μm using a series of stacked sieves.

Comparative Example A: Heptane (348 ml), toluene (188 ml) and 1.4 ml ofpolymer stabilizer solution were added to a 1 L Mortonized round bottomflask equipped with an overhead stirrer, thermocouple, reflux condenser,and nitrogen gas inlet. The overhead stirrer was adjusted to a stir rateof approximately 360 rpm and the reaction flask was heated to 35° C.under a slow nitrogen gas purge. MBA (13.3 g) andN-acryloylmethylalanine (AMA, 0.7 g) were added to a 250 ml Erlenmeyerflask equipped with a stir bar. Isopropyl alcohol (65 ml) and 47 ml ofwater were added to dissolve the solids. Ethylene glycol (25 ml) wasthen added. Upon dissolution of all solids, sodium persulfate (0.55 g in3 ml water) was added to the stirred solution. The aqueous phase wasadded to the organic phase and mixed until the reaction reached 35° C.Tetramethylethylenediamine (0.55 ml) was added to initiate the reaction.The polymerization reaction was stirred for 2 hrs until particlesformed.

The particles were course filtered and washed twice with acetone (250 mleach), twice with methanol (250 ml each), and then twice with acetone(250 ml each). The resulting particles were transferred to a 500 mlErlenmeyer flask. Acetone (300 ml) was added to suspend the particles.The suspended particles were sonicated for approximately 15 minutes, andthen filtered. The resulting particles were classified to a meanparticle size of approximately 65 μm using a series of stacked sieves.

Following preparation, the resulting particles were washed thoroughlywith acetone and suspended in 500 ml dry dimethylsulfoxide. To thisslurry was added acetic anhydride (25 ml) and triethylamine (2 ml). Theparticles were agitated by rocking for an hour, filtered, and thenwashed extensively with acetone and MTBE. The resultantazlactone-functional reactive beads were suspended in an aqueous 1 Msolution of benzylamine and allowed to react for 1 hour. The beads werethen filtered and washed extensively with distilled water.

Experimental Methods

Preparation of Chromatography Column: Chromatography columns wereprepared by slurry packing the exemplary particles into a 3.0 mm×150 mmglass tube supplied by Omifit, Cambridge, CB1 3HD England. Porous Teflonfrits (25 μm average pore size, Small Parts, Inc., Miami Lakes, Fla.)were placed at both ends of the tube to form a chromatography column.

Preparation of Chromatography System: The chromatography column wasassembled in an FPLC (fast protein liquid chromatograph, obtained underthe trade designation “AKTA FPLC”, GE Healthcare, Uppsala, Swedenequipped with a UV detector and a conductivity detector.

Protein Analysis: The chromatography column in the chromatography systemwas equilibrated with a mobile phase of 50 mM (millimolar) sodiumphosphate, pH 7 with 1.0 M sodium citrate at a flow rate of 0.088mL/min. 200 μL (microliter) of a solution containing 0.30 mg(milligram)/ml myoglobin (from Sigma-Aldrich Chemical Company;Milwaukee, Wis.), 0.24 mg/ml β-lactoglobulin (from USB Corporation,Cleveland, Ohio), 0.11 mg/ml lysozyme (from Sigma-Aldrich ChemicalCompany), and 0.14 mg/ml bovine serum albumin (BSA) (from Sigma-AldrichChemical Company) in 50 mM sodium phosphate, pH 7 with 1.0 M sodiumcitrate was injected onto the chromatography column. A gradient elution(40 column volumes) from the initial buffer condition (high salt) to 50mM sodium phosphate, pH 7 (low salt) was applied. Using UV detection,the eluent was monitored at a 280 nm (nanometer) wavelength.

Dynamic Binding Capacity Analysis: The chromatography column in thechromatography system was equilibrated with a mobile phase of 0.6Msodium citrate, pH 6.0. A solution of 2.3 mg/mL of human IgG (hIgG fromEquitech, Kerrville, Tex.) in 0.6M sodium citrate at a pH 6.0 was pumpedthrough the chromatography column at a flow rate of 170 cm/hr. Using UVdetection, the eluent was monitored at a 280 nm wavelength. The 280 nmabsorbance was correlated with IgG concentration. The dynamic bindingcapacity (DBC) was determined by monitoring the IgG breakthrough (10% ofmaximum protein concentration eluting from the column).

Analysis Using Particles

Example 17: The particles prepared in Example 12 were packed into a tubeto form a chromatography column using the method as described above andan analysis was preformed using the Protein Analysis method as describedabove. Shown in FIG. 1 is the chromatogram.

Example 18: The particles prepared in Example 13 were packed into a tubeto form a chromatography column using the method as described above andan analysis was preformed using the Protein Analysis method as describedabove. Shown in FIG. 1 is the chromatogram.

Shown in FIG. 1 is an overlay of the chromatogram from Example 17 (usingthe particles prepared in Example 12) and Example 18 (using theparticles prepared in Example 13). The particles prepared in Examples 12and 13 were prepared using the same monomer composition and reactionconditions, however, the PEG additive had an effect on the overallhydrophobicity of the particles. As shown in FIG. 1, the particleprepared in Example 12 with PEG 2,000 was more hydrophobic (elution ofproteins required a lower salt buffer) than the particle prepared inExample 13 with PEG 6,000. The resolution of BSA was comparable in bothExamples 17 and 18, however, the largest eluting peak (a co-elution ofB-lactoglobulin and lysozyme) was sharper in Example 17 (the particleprepared with PEG 2,000).

Example 19: The particles prepared in Example 12 were packed into a tubeto form a chromatography column using the method as described above andan analysis was preformed using the Dynamic Binding Capacity method asdescribed above. Shown in FIG. 2 is the breakthrough curve.

Example 20: The particles prepared in Example 13 were packed into a tubeto form a chromatography column using the method as described above andan analysis was preformed using the Dynamic Binding Capacity method asdescribed above. Shown in FIG. 2 is the breakthrough curve.

Comparative Example B: An aromatic HIC media of highly cross-linked 90μm agarose beads derivatized with phenyl groups via an ether linkagesold under the trade designation “PHENYL SEPHAROSE 6 FAST FLOW (LOWSUB)” commercially available from GE Healthcare, Chalfont St. Giles,United Kingdom were packed into a tube to form a chromatography columnusing the method as described above and an analysis was preformed usingthe Dynamic Binding Capacity method as described above. Shown in FIG. 2is the breakthrough curve.

Comparative Example C: An aromatic HIC media of highly cross-linked 90μm agarose beads derivatized with phenyl groups via an ether linkagesold under the trade designation “PHENYL SEPHAROSE 6 FAST FLOW (HIGHSUB)” commercially available from GE Healthcare, Chalfont St. Giles,United Kingdom were packed into a tube to form a chromatography columnusing the method as described above and an analysis was preformed usingthe Dynamic Binding Capacity method as described above. Shown in FIG. 2is the breakthrough curve.

FIG. 2 is an overlay of the breakthrough curves for Examples 19 and 20and Comparative Examples B and C. From the breakthrough curves shown inFIG. 2 the breakthrough was calculated to be as follows: Example 19=58mg/mL, Example 20=49 mg/mL, Comparative Example B=38 mg/mL, andComparative Example C=54 mg/mL. The calculated breakthrough indicatesthat the particles of Example 19 had the most adsorbance of IgG,followed by Comparative Example C, Example 20, and then ComparativeExample B. Also shown in FIG. 2 is the profile of the breakthrough foreach of the examples, which indicates how the IgG is being adsorbed bythe particles/beads. Examples 19 and 20 provided a sharper breakthroughcurve (steepness of the exponential growth) than Comparative Example C.Comparative Example B not only had the lowest DBC at 10% breakthrough,but also did not show a flat baseline, indicating that there wasinconsistent binding of IgG to the beads. The amount of phenyl per ml ofthe particles/beads shown in FIG. 2 are as follows: Example 19=14 μmol(micromole) phenyl/ml particle (calculated based on the amount of phenylmonomer used to make the particle and the swell volume of the particle),Example 20=13 μmol phenyl/ml particle (calculated based on the amount ofphenyl monomer used to make the particle and the swell volume of theparticle), Comparative Example B=25 μmol phenyl/ml particle (taken fromproduct literature), and Comparative Example C=50 μmol phenyl/mlparticle (taken from product literature). Based on the data shown inFIG. 2, the particles according to the present disclosure havecomparable or better DBC than Comparative Example C, while havingsubstantially less phenyl groups per ml of particle.

Example 21: The particles prepared in Example 14 were packed into a tubeto form a chromatography column using the method as described above andan analysis was preformed using the Protein Analysis method as describedabove. Shown in FIG. 3 is the chromatogram.

Example 22: The particles prepared in Example 15 were packed into a tubeto form a chromatography column using the method as described above andan analysis was preformed using the Protein Analysis method as describedabove. Shown in FIG. 3 is the chromatogram.

Shown in FIG. 3 is an overlay of the chromatogram from Example 21 (usingthe particles prepared in Example 14) and Example 22 (using theparticles prepared in Example 15). The particles prepared in Examples 14and 15 were prepared using the same monomer composition and reactionconditions, however, the PEG additive had an effect on the overallhydrophobicity of the particles. As shown in FIG. 3, the particleprepared in Example 14 with PEG 6,000 was more hydrophobic than theparticle prepared in Example 15 with PEG 10,000. However, the particleprepared in Example 15 with PEG 10,000 was able to resolve BSA fromB-lactoglobulin and lysozme, whereas the particle prepared in Example 14with PEG 6,000 showed no resolution of the BSA peak.

Example 23: The particles prepared in Example 15 were packed into a tubeto form a chromatography column using the method as described above andan analysis was preformed using the Dynamic Binding Capacity method asdescribed above. The particle had a calculated DBC of 34 mg/ml. Thisvalue is less than the DBC values for the particles prepared with AAm asa comonomer (as shown in Examples 19 and 20 above).

Example 24: The particles prepared in Example 16 were packed into a tubeto form a chromatography column using the method as described above andan analysis was preformed using the Protein Analysis method as describedabove. Shown in FIG. 4 is blow-up of the chromatogram comprising the IgGpeak.

Comparative Example D: The particles prepared in Comparative Example Awere packed into a tube to form a chromatography column using the methodas described above and an analysis was preformed using the ProteinAnalysis method as described above. Shown in FIG. 4 is blow-up of thechromatogram comprising the IgG peak.

Shown in FIG. 4 is an overlay of the IgG peak from Example 24 andComparative Example D. The particles used in Example 24 and ComparativeExample D have the same particle composition; azlactone linkagescovalently bonding acrylamide particles with the benzylamine hydrophobicgroup. Thus, one would expect them to have the same retention time. Asshown in FIG. 4, the IgG peak in Example 24 is retained less than inComparative Example D. Although not wanting to be bound by theory, theincreased retention of IgG in Comparative Example D is speculated to bedue to the presence of a small amount of negative charge generated byhydrolysis of the azlactone during the reaction with benzylamine.Further, as shown in FIG. 4, the IgG peak in Comparative Example D isbroader than that in Example 24, which is also speculated to beindicative of a mixed interaction (e.g. hydrophobic interactions and ionexchange interactions) of the IgG with the stationary phase.

Various modifications and alterations to this disclosure will becomeapparent to those skilled in the art without departing from the scopeand spirit of this disclosure. It should be understood that thisdisclosure is not intended to be unduly limited by the illustrativeembodiments and examples set forth herein and that such examples andembodiments are presented by way of example only with the scope of thedisclosure intended to be limited only by the claims set forth herein asfollows.

We claim:
 1. A method of separating a biomolecule in an aqueous mixture,the method comprising: (a) obtaining a separation vessel containingseparation media, wherein the separation media comprises a poroussupport with a hydrophobic monomer grafted thereon, the hydrophobicmonomer having the structure:CH₂═CR⁴C(O)NHC(R¹R¹)(C(R¹R¹))_(n)C(O)XR³ wherein n is an integer of 0 or1; R¹ is independently selected from at least one of: a hydrogen atom,alkyls, aryls, and alkylaryls, wherein the alkyls, aryls, and alkylarylshave a total of 10 carbon atoms or less; R³ is a hydrophobic groupselected from at least one of alkyls, aryls, alkylaryls and ethers,wherein the alkyls, aryls, alkylaryls and ethers have a total number ofcarbon atoms ranging from 4 to 30; R⁴ is H or CH₃; and X is O or NH;wherein the hydrophobic monomer is derived from an amine or an alcohol(HXR³) that has a hydrophilicity index of 25 or less; and (b) passingthe aqueous mixture through the separation vessel thereby separating thebiomolecule.
 2. The method of claim 1, wherein R¹ is independentlyselected from at least one of: methyl, ethyl, phenyl, or combinationsthereof.
 3. The method of claim 1, wherein R³ is selected from at leastone of: benzyl, phenethyl, phenoxyethyl, phenylpropyl, butyl, pentyl,hexyl, octyl, dodecyl, octadecyl, phenylbutyl, or combinations thereof.4. The method of claim 1, wherein R³ is selected from: benzyl,phenethyl, phenoxyethyl, phenylpropyl, butyl, pentyl, hexyl, octyl,phenylbutyl, or combinations thereof.
 5. The method of claim 1, whereinn is
 1. 6. The method of claim 1, wherein the hydrophobic monomer hasthe following structure:CH₂═CR⁴C(O)NHC(R²R²)(C(R¹R¹))_(n)C(O)NHR³ wherein n is an integer of 0or 1; wherein each R² is independently selected from: alkyls, aryls, andalkylaryls, wherein the alkyls, aryls, and alkylaryls have a total of 10carbon atoms or less; each R¹ is independently selected from: alkyls,aryls, and alkylaryls, wherein the alkyls, aryls, and alkylaryls have atotal of 10 carbon atoms or less; R³ is a hydrophobic group selectedfrom: alkyls, aryls, alkylaryls and ethers, wherein the alkyls, aryls,alkylaryls and ethers have a total number of carbon atoms ranging from 4to 30; and R⁴ is H or CH₃.
 7. The method of claim 6, wherein R² isselected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,n-pentyl, n-hexyl, n-heptyl, n-octyl, and ethylhexyl.
 8. The method ofclaim 6, wherein R² is selected from: methyl or ethyl.
 9. The method ofclaim 6, wherein R² is the same.
 10. The method of claim 6, wherein nis
 1. 11. The method of claim 1, wherein the hydrophobic monomer has astructure selected from at least one of:CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)NH(CH₂)₄C₆H₅;CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)O(CH₂)₄C₆H₅;CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)NHCH₂C₆H₅;CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)NH(CH₂)₂C₆H₅;CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)NH(CH₂)₂OC₆H₅;CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)NH(CH₂)₃C₆H₅;CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)NH(CH₂)₃CH₃;CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)NH(CH₂)₅CH₃;CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)NH(CH₂)₇CH₃;CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)NH(CH₂)₁₁CH₃;CH₂═CHC(O)NHC(CH₃)(CH₃)C(O)NH(CH₂)₁₇CH₃; or combinations thereof. 12.The method of claim 1, wherein the porous support is selected from awoven web, a nonwoven web, a fibrous web, a microporous membrane, amicroporous film, and combinations thereof.
 13. The method of claim 1,wherein the biomolecule is selected from at least one of: proteins,antibodies, fusion proteins, vaccines, peptides, enzymes, DNA, or RNA.14. The method of claim 1, wherein the biomolecule is abiomacromolecule.
 15. The method of claim 1, wherein the aqueous mixturecomprises a buffer.
 16. The method of claim 1, wherein the aqueousmixture is a biological sample.